Hydrogen uptake by Robinia pseudoacacia nodules

Summary Hydrogen uptake is thought to increase the efficiency of nitrogen fixation by recycling H₂ produced by nitrogenase that would otherwise be lost by diffusion. Here we demonstrate the capacity of eight Rhizobium strains to take up molecular hydrogen. Uptake by nodule homogenates from Robinia pseudoacacia was measured amperometrically under nitrogenase repression. Markedly lower activities were found than in soybean nodules. In addition hydrogenase activity was detected by the ability of bacteroids to reduce methylene blue in the presence of hydrogen. It was demonstrated that hydrogenase structural genes are present in the black locust symbiont, Rhizobium sp. strain R1, using hybridization with a plasmid, which contained hydrogenase genes from R. leguminosarum bv. viceae.     

基于光周期和温度的物候模型对华北平原刺槐始花期的模拟

光周期和温度是影响木本植物开花的两大关键气象因素。基于我国华北平原1963—2018年57个站点的刺槐始花期实测数据,构建了6种基于气温或光周期驱动的春季物候过程模型(积温模型、光周期模型、光周期-温度顺序模型、温度-光周期顺序模型、光周期-温度平行模型、光周期-温度乘法模型),并进行了参数率定和模型比较优选,以期明确刺槐开花的主导驱动因子以及潜在的光温作用机制。根据内部模拟的赤池信息准则(AIC)判定光周期-温度顺序模型的表现最佳,模拟值与观测值的相关系数(r)为0.86,均方根误差(RMSE)为4.81d,纳什效率系数(NSE)达到0.74,说明刺槐始花期的发生同时受到光周期和温度的作用。同时,在华北平原14个代表性站点上,光周期-温度顺序模型的表现普遍优于均值模型。基于光周期-温度顺序模型的参数率定结果,以下限温度6.5℃和下限日长4.5h来统计刺槐开花的实际有效积温量和实际有效日长累积量的年际变化情况,结果表明56年来刺槐开花的实际有效积温量呈现显著递增趋势,平均每10年增加4.5℃·d(P<0.05),而实际有效日长累积量却呈现极显著递减趋势,平均每10年减少23.9h·d(P<0.01),这说明在气候变暖背景下,刺槐开花的热量需求会更快速完成,引起刺槐开花日期的提前,但自然光周期的年内变化只与地理位置有关,日长累积需求则需要更长的时间完成,因此会在一定程度上抑制春季升温引起的开花日期提前。     

Formation of heartwood substances in the stem of Robinia pseudoacacia L.

Summary The activities of two key enzymes in flavonoid biosynthesis, phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) and chalcone synthase (CHS, E.C. 2.3.1.74) were determined in the trunkwood of Robinia pseudoacacia L. The trees under investigation were cut at different times of the year (September, November, January and April). At all times PAL is active, both in the youngest wood layer (the outermost growth ring) and at the sapwood heartwood boundary. On the other hand, CHS is active exclusively in the vicinity of the heartwood boundary. The results indicate that PAL is involved both in the formation of lignin (outermost annual ring), and in flavonoid biosynthesis (heartwood boundary). Highest activity of both PAL and CHS could be measured at the sapwood heartwood boundary in the tree felled in November, indicating that heartwood formation was occurring mainly at that time. The flavonoids accumulated in the heartwood are obviously formed in situ and seem to be transported only to a minor extent — if at all — via the phloem and the ray cells to the heartwood.     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

Nitrification potentials in early successional black locust and in mixed hardwood forest stands in the southern Appalachians,USA

Soil nitrogen mineralisation and nitrification potentials, and soil solution chemistry were measured in black locust (Robinia pseudo-acacia L.), in pine-mixed hardwood stands on an early successional watershed (WS6), and in an older growth oak-hickory forest located on an adjacent, mixed hardwood watershed (WS14) at Coweeta Hydrologic laboratory, in the southern Appalachian mountains, U.S.A. Nitrification potentials were higher in black locust and pine-mixed hardwood early successional stands than in the oak-hickory forest of the older growth watershed. Ammonification rates were the main factor controlling nitrification in the early successional stands. There was no evidence of inhibition of nitrification in soils from the older growth oak-hickory forest site.Within the early successional watershed, black locust sites had net mineralisation and nitrification rates at least twice as high as those in the pine mixed-hardwood stands. Concentrations of exchangeable nitrate in the soil of black locust stands were higher than in pine-mixed hardwoods at 0–15 cm in March and they were also higher at 0–15, 16–30 and 31–45 cm depth in the black locust dominated sites in July. Soil solution nitrate concentrations were higher under black locust than under pine-mixed hardwoods. Areas dominated by the nitrogen fixing black locust had greater nitrogen mineralisation and nitrification rates, resulting in higher potential for leaching losses of nitrate from the soil column in the early successional watershed.     

Phenotypic,genomic and phylogenetic characteristics of rhizobia isolated from root nodules of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> (black locust) growing in Poland and Japan

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98–99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771–961 kb megaplasmids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A non-destructive field assay for soybean nitrogen fixation by acetylene reduction

Summary A system for employing open-ended root chambers to measurein situ acetylene reduction rates under field conditions is described. Gas mixtures containing about 2 mbar acetylene were continuously flowed through the chambers providing a continuous record of acetylene reduction. These chambers have been used to measure acetylene reduction rates of soybeans during three growing seasons. The system has proved to be reliable with a high degree of precision. The large amount of plant-to-plant variability observed in N₂ fixation research has been confirmed by the data collected with this system. However, such variability in physiological studies can be reduced by using a non-destructive system to compare the response of an individual plant with its rates before treatment.     

丛枝菌根真菌对刺槐幼苗机械损伤响应机制的初步研究

通过对刺槐幼苗每隔3d剪去1片叶片造成持续机械损伤,测定了0~138h抗氧化酶活性变化及刺槐幼苗生长情况;同时使用孔径25μm尼龙网设置三室根箱隔网系统,测定了供体瞬时机械损伤后受体的抗氧化酶活性的持续变化,探讨接种丛枝菌根真菌刺槐幼苗对持续及瞬时机械损伤后的响应机制,以及菌根菌丝桥对刺槐幼苗机械损伤信号的传递特征。结果表明:在持续机械损伤胁迫下,接种丛枝菌根真菌能够促进刺槐幼苗的根系生长、提高幼苗的成活率,接种丛枝菌根真菌的刺槐幼苗成活率及根系鲜重比对照分别增加15.38%和23.52%。瞬时机械损伤后0、48、90、114、138h刺槐幼苗的苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)活性都呈现先增加后降低的趋势,均在90h达到最大值,并且菌根化幼苗的PAL和POD活性显著高于未菌根化幼苗。瞬时机械损伤后,菌根菌丝桥能够介导刺槐幼苗间相关信号的传递,从而引起菌根化受体刺槐幼苗的PAL和POD活性表现出与供体机械损伤幼苗相同的变化趋势。     

Nitrate reductase activity in leaves and roots ofAlnus glutinosa (L) Gaertner

Summary Thein vivo nitrate reductase activity (NRA) was determined inAlnus glutinosa plants grown nonsymbiotically on ammonium, nitrate, a combination of both, or symbiotically with atmospheric nitrogen as the only nitrogen source. Root NRA was absent when ammonium or atmospheric nitrogen was the nitrogen source. With nitrate in the culture solution the roots showed a high NRA. However, the leaf NRA behaved quite differently: with negligible activities on all nitrogen sources except atmospheric nitrogen. The foliar NRA measured, however, is likely not due to the activity of the plant but of microbial origin. Methods commonly used to facilitate produced nitrite to leak out of the tissue, such as addition of propanol and cutting the plant material, did not increase the nitrite release from the leaves. A turbidity developed when testing the samples for nitrite which was positively correlated with the NRA. Populations of microorganisms in the phyllosphere did not differ between the nutritional treatments. Bacteria, able to grow on a low-nitrogen medium, were present on the leaves. Nitrifiers could not be detected. The bacteria on the leaves appear to produce nitrite when incubated with leaf material. Grassland Species Research Group, Publication no. 106     

Aspects of nitrogen fixation and denitrification byAzospirillum

Summary Model experiments were performed to investigate the nitrogen fixation (C₂H₂ reduction) and denitrification (N₂O formation) capabilities ofAzospirillum spp. in association with wheat. Plants and bacteria were grown together for a week and then assayed for activities. This association performed C₂H₂ reduction or N₂O formation, depending on the concentrations of nitrate and oxygen in the vessels. Both activities depended on theAzospirillum strains used. The newly isolatedAzospirillum amazonense strains Y1 and Y6 showed significant C₂H₂ reduction and low N₂O formation in association with wheat under the conditions employed and are possibly useful in practice. A cell-free preparation fromAzospirillum brasilense Sp 7 possessed a cytochrome cd type dissimilatory nitrite reductase.     

4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

Host-genetic control of nitrogen fixation in the legume-Rhizobium symbiosis: complication in the genetic analysis due to maternal effects

Summary In pea cv. Afghanistan a recessive gene sym 6 prevents the full expression of nitrogenase activity in root nodules, induced byRhizobium leguminosarum strain F 13. In contrast, nitrogenase activity is fully expressed in pea cv. Iran. A comparison of the reciprocal hybrids of these two plants showed that the size of the plant was determined by the mother plant (maternal effect). Therefore the shoot weight and the total amount of nitrogen fixed are not suitable as parameters for a genetic analysis. The % nitrogen of the shoot and the specific activity of the nodules per gram of nodules are more reliable, but for practical purposes the specific activity of the nodules expressed per gram of shoot tissue can be used.     

黄土高原南北样带刺槐林土壤碳、氮、磷生态化学计量特征

为了解黄土高原南北样带刺槐林土壤碳（C）、氮（N）、磷（P）化学计量特征,采集了黄土高原南北样带上12个典型样点的刺槐林土壤,测定了土壤有机碳（SOC）、全氮（TN）、全磷（TP）含量并分析其生态化学计量特征。结果表明：刺槐林土壤SOC、TN、TP含量均随降水量降低而降低,且土壤SOC与TN的空间变化具有一致性;土壤SOC、TN、TP及其生态化学计量比与降雨量呈显著正相关（P < 0.05）,且TN、C：P、N：P与土壤含水量也呈显著正相关（P < 0.05）。     

The seed lectins of black locust (robinia pseudoacacia) are encoded by two genes which differ from the bark lectin genes

Two lectins were isolated from Robinia pseudoacacia (black locust) seeds using affinity chromatography on fetuin-agarose, and ion exchange chromatography on a Neobar CS column. The first lectin, R. pseudoacacia seed agglutinin I, referred to as RPsAI, is a homotetramer of four 34 kDa subunits whereas the second lectin, referred to as RPsAII, is composed of four 29 kDa polypeptides. cDNA clones encoding the polypeptides of RPsAI and RPsAII were isolated and their sequences were determined. Both polypeptides are translated from mRNAs of ca. 1.2 kb encoding a precursor carrying a signal peptide. Alignment of the deduced amino acid sequences of the different clones indicates that the 34 and 29 kDa seed lectin polypeptides show 95% sequence identity. In spite of this striking homology, the 29 kDa polypeptide has only one putative glycosylation site whereas the 34 kDa subunit has four of these sites. Carbohydrate analysis revealed that the 34 kDa possesses three carbohydrate chains whereas the 29 kDa polypeptide is only partially glycosylated at one site. A comparison of the deduced amino acid sequences of the two seed and three bark lectin polypeptides demonstrated unambiguously that they are encoded by different genes. This implies that five different genes are involved in the control of the expression of the lectins in black locust.Abbreviations LECRPAs cDNA clone encoding Robinia pseudoacacia seed lectin - LoLI Lathyrus ochrus isolectin I - PsA Pisum sativum agglutinin - RPbAI Robinia pseudoacacia bark agglutinin I - RPbAII Robinia pseudoacacia bark agglutinin II - RPsAI Robinia pseudoacacia seed agglutinin I - RPsAII Robinia pseudoacacia seed agglutinin II     

黄土丘陵区刺槐与油松人工林生态系统生态化学计量特征

为阐明不同人工林生态系统间生态化学计量特征的差异,采用野外采样与室内分析相结合的方式分析了陕北黄土丘陵区落叶阔叶树种刺槐和常绿针叶树种油松人工林乔木、灌草、枯落物和土壤(土层深度0—100cm)C、N、P化学计量特征。结果表明:1)刺槐乔木各器官(叶、枝、干、皮、根)C含量显著低于油松,但N和P含量显著高于油松。因此,油松的C∶N和C∶P显著大于刺槐,而N∶P小于刺槐。2)刺槐林下枯落物N和P含量显著高于油松,但C含量显著小于油松。此外,油松林下枯落物C∶N(70.21)大于刺槐林下枯落物C∶N(19.71),说明油松林下枯落物分解较慢,有利于养分的存储。3)刺槐和油松人工林土壤C、N含量均随土壤深度增加而减少,P含量则基本保持不变。刺槐人工林土壤中C含量低于油松,N、P含量在两者之间无显著差异。4)刺槐人工林内乔灌草叶、枯落物与土壤C、N、P及其计量比的相关性多集中在10—20、20—30cm土层,而油松林中各组分与土壤营养元素的相关性相对较小,其中20—30cm土层中无显著相关性,说明相比刺槐人工林而言,油松人工林内土壤层N、P供应量对植物叶片N、P含量影响不显著。本研究为深入了解黄土丘陵区生态系统养分耦合循环机制奠定了基础,同时也为黄土丘陵区的植被恢复工作提供了一定的指导意义。     

Co-evolution of the legume-Rhizobium association

Summary A number of examples is given demonstrating the co-existence of pea genotypes and their specific Rhizobium, strains isolated within the same region.R. leguminosarum strains compatible with the cultivated pea have a narrow symbiotic range and they are widely distributed in European soils. This is presumably due to the narrow genetic base of the cultivated pea and its wide-spread cultivation in European soils. Rhizobium strains capable of nodulating a primitive pea line from Afghanistan were only found in soils of the Middle East and Central Asia. A more restricted distribution of specific Rhizobium strains was found for fulvum peas from Israel. Rhizobium strains effective with the fulvum pea were found in Israeli soils. A good example of co-evolution due to geographical isolation was found in south Turkey. Here a pea line was found which can form an effective symbiosis with local Rhizobium strains but not with strains from other parts of Turkey.     

Relationship between growth,nitrogen fixation and assimilation in a legume (Medicago sativa L.)

Summary Symbiotic N₂ fixation, NO ₃ ⁻ assimilation and protein accumulation in the shoots were measured simultaneously in alfalfa (Medicago sativa L.) grown in the field or in pots, in order to study how the balance between the two modes of nitrogen nutrition could be influenced by agronomic factors, such as harvest, mineral nitrogen supply and drought stress. During periods of rapid growth, fixation and assimilation may function simultaneously; they are antagonistic at the beginning and at the end of the growth cycle, when the nitrogen requirement of the plant is lower. When nitrogen nutrition does not limit growth, mineral nitrogen supply favours assimilation at the expense of fixation, but does not modify the amount of nitrogen accumulated, which is adjusted to the growth capacity of the plant. After cutting, nitrate assimilation compensated for the decrease in fixation and supplied the plant with the nitrogen required by the regrowth, the proliferation of which determined the fixation recovery. Drought stress decreased N₂ fixation much more than NO ₃ ⁻ assimilation. The latter made growth recovery possible when water supply conditions became normal again. These results suggested the existence of an optimum level of nitrate assimilation, which differed depending on the age of the plants and allowed both maximum growth and fixing activity.     

Development of the nitrogen fixing apparatus in the legumes,Centrosema pubescens Benth., and Vigna unguiculata L. Walp.

The sequence of events leading up to the establishment of symbiotic nitrogen-fixation were studied in two tropical legumes, Centrosema pubescens Benth, and Vigna unguiculata L. Walp. Parameters measured included fresh and dry weights, chlorophyll and leghaemoglobin contents, as well as the activities of NADH-nitrate reductase (EC 1.6.6.1), and nitrogenase (nitric-oxide reductase-EC 1.7.99.2) in plants that were inoculated with suitable rhizobia or which were watered with potassium nitrate. Dry weight and photosynthetic activity of both species followed the sigmoidal pattern which is characteristic of most plants. Growth was little different in either a qualitative or quantitative sense whether nitrogen was supplied as nitrate or through dinitrogen fixation. Although the biochemical sequence of events was dependent on the limiting sensitivities of the individual assays used, the data suggest that nitrate reductase is the first measurable enzymatic activity in the nodules (and roots), followed by acetylene reduction and leghaemoglobin in that order. It is possible therefore, that low levels of symbiotic nitrogen fixation occur in the nodules in the absence of leghaemoglobin. Nitrate reductase activity in C. pubescens nodules was negatively exponentially correlated with nitrogenase activity of the same nodules, suggesting a changing metabolism in old nodules. These data are discussed in terms of environmental and physical factors known to control nitrogen fixation.     

不同林龄刺槐林土壤团聚体化学计量特征及其与土壤养分的关系

土壤团聚体化学计量特征分析可以为土壤养分的评价提供依据,对陕北黄土丘陵区20 a、25 a、40 a、50 a刺槐林土壤团聚体有机碳、全氮、全磷化学计量比及其与土壤有机碳、全氮、全磷化学计量比的相关性采用逐步回归分析方法进行了分析。结果表明:随着林龄的增加,刺槐林各粒径土壤团聚体有机碳、全氮含量及其有机碳、全氮、全磷化学计量比显著增加(P0.05),均表现为在0—20 cm土层高于20—40 cm土层,而刺槐林土壤团聚体全磷含量变化较小;相同林龄刺槐林在0—20 cm和20—40cm土层中0.25—2 mm粒径土壤团聚体有机碳、全氮、全磷含量及其化学计量比最高。刺槐林0.25—2 mm粒径团聚体对土壤原土有机碳、全氮含量及其有机碳、全氮、全磷化学计量比有显著影响。营造刺槐林对各粒径土壤团聚体全效养分分配及其平衡关系存在积极的影响,主要体现在0.25—2 mm粒径土壤大团聚体中,通过影响0.25—2 mm粒径团聚体提高了土壤全效养分的供应和保持能力。